| 1. |
- Hult, Andreas, 1981-
(författare)
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Towards a detailed understanding of the red blood cell storage lesion : and its consequences for in vivo survival following transfusion
- 2015
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Red blood cells (RBCs) are vital for oxygen delivery to tissues and constitute the vast majority of all cells in blood. After leaving the red bone marrow as mature cells, RBCs have a lifespan of approximately 120 days before they are removed from the circulation by macrophages, mainly in the spleen and liver. RBC transfusion is a common therapy in modern healthcare. Major surgery, numerous cancer treatments and other, often lifesaving, interventions would be unthinkable without available blood supply. For this reason, hospitals store donated RBCs in blood banks.The metabolic and structural changes that occur during prolonged storage of RBCs (the storage lesion) have been studied in detail in vitro and include oxidative stress, a reduction in glycolysis, increased membrane rigidity and shedding of microparticles from the RBC membrane. Stored RBCs share several features of senescent RBCs, but also with RBCs undergoing an apoptotic-like process called eryptosis. A consequence of the storage lesion is the fact that as much as 25% of stored RBCs could be rapidly removed from the circulation within 24 hours after transfusion. The mechanisms behind this rapid macrophage-mediated recognition and removal of stored RBCs, and its immunological consequences, remain largely unknown. Therefore, the aims of this thesis were to investigate if cryopreserved human RBCs induced an inflammatory response following autologous transfusion into healthy volunteers, and to further understand the mechanisms behind macrophage recognition of stored RBCs in vitro and in vivo.Autologous transfusion of two units of cryopreserved RBCs into healthy human recipients was found to be associated with an increased extravascular RBC elimination already at 2 hours after transfusion. However, there were no signs of an increased production of any of the investigated pro-inflammatory cytokines, indicating that an increase in the destruction of RBCs per se did not induce an inflammatory response.Eryptosis is a form of induced RBC death associated with an increased cytoplasmic Ca2+ uptake. We found that a subset of human RBCs increased their Ca2+ permeability during prolonged storage at +4°C. Using a murine model, to further understand how RBCs with an increased Ca2+ permeability were eliminated by phagocytic cells in the spleen, it was found that such RBCs were taken up by marginal zone macrophages and dendritic cells (DCs) in a manner distinct from that of naturally senescent RBCs. The DC population particularly efficient in this process expressed CD207 and are known for their ability to promote immunological tolerance. Eryptotic cell uptake was not regulated by the phagocytosis-inhibitory protein CD47 on the RBCs.To investigate how RBCs damaged during liquid storage are recognized and taken up by macrophages, a model to store and transfuse murine RBCs was developed. This storage model generated murine RBCs with several characteristics similar to that of stored human RBCs (i.e. loss of ATP, formation of RBC microparticles and rapid clearance of up to 35% of the RBCs during the first 24 h after transfusion). In vitro phagocytosis of human as well as murine stored RBCs was serum dependent and could be inhibited by blocking class A scavenger receptors using fucoidan or dextran sulphate.In conclusion, the findings of this thesis contribute to further understanding how changes inflicted to RBCs during storage direct the fate of these cells in their interaction with cells of the immune system after transfusion. The observation of an increased Ca2+ permeability of stored RBCs, and the possible recognition of such cells by tolerance-promoting DCs, in combination with the findings that class A scavenger receptors and serum factors may mediate recognition of stored RBCs, may result in novel new directions of research within the field of transfusion medicine.
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| 2. |
- Kolan, Shrikant S, 1983-
(författare)
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Defining the role of CD47 and SIRPα in murine B cell homeostasis
- 2015
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- B cell development is a highly organized process, which commences in the fetal liver during embryogenesis and in the bone marrow (BM) after birth. Surface IgM+ immature B cells emigrate from the BM via the blood stream to the spleen and finally differentiate into conventional mature follicular B (FoB) cells and marginal zone (MZ) B cells. Conversely, some sIgM+ immature B cells can also mature into IgD+ FoB cells in the BM.The ubiquitously expressed cell surface glycoprotein CD47 and its receptor signal regulatory protein α (SIRPα) are members of the immunoglobulin superfamily. Both individually and upon their interaction, CD47 and SIRPα have been found to play important role in the homeostasis of T lymphocytes or CD8 conventional dendritic cells (cDCs) in secondary lymphoid organs. However, their role in regulating B cell homeostasis has remained unknown.The present study describes important roles of CD47 and SIRPα in B cell homeostasis. Lack of SIRPα signaling in adult SIRPα mutant (MT - cytoplasmic domain deletion) mice resulted in an impaired B cell maturation in the BM and spleen, which was also reflected in the blood. In the BM and spleen of SIRPα MT mice, reduced numbers of semi-mature IgD+IgMhi follicular type-II (F-II) and mature IgD+IgMlo follicular type-I (F-I) B cells were observed, while earlier BM B cell progenitors or splenic transitional B cells remained unaltered. In SIRPα MT mice, maturing B cells in BM and spleen were found to express higher levels of the pro-apoptotic protein BIM and contained an increased level of apoptotic cells.In contrast to that for FoB cells, the splenic MZ B cell population was increased with age in SIRPα MT mice without showing an increased level of activation markers. Immunohistochemical analysis revealed an increased follicular localization of MZ B cells in the spleens of SIRPα MT mice. In addition, MZ macrophages and marginal metallophilic macrophages were not restricted to their normal position in SIRPα MT spleens. Interestingly, CD47-deficient (CD47-/-) mice mimicked the FoB cell phenotype observed in SIRPα MT mice and had a reduced number of FoB cells in the BM, blood and the spleen at 56 months of age, but not in younger mice. Similar to SIRPα MT mice, CD47-/- mice also displayed an increased number of splenic MZ B cells. Sera form both mouse strains did not show any signs of an increased production of autoantibodies or antinuclear antigens.BM reconstitution experiments identified a requirement for non-hematopoietic SIRPα signaling for normal B cell maturation in the BM and to maintain normal numbers and retention of MZ B cells in the splenic MZ. On the contrary, hematopoietic SIRPα signaling appeared to be important for FoB cell maturation in the spleen. Interestingly, hematopoietic SIRPα was required for normal MZ retention of MZ macrophages while normal distribution of metallophilic macrophages required nonhematopoietic SIRPα signaling. Collectively, these findings revealed an important role of CD47 and of SIRPα signaling in B cell homeostasis in different lymphoid organs.
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| 3. |
- Koskinen, Cecilia, 1979-
(författare)
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CD47–SIRPα : an interaction of importance for bone cell differentiation
- 2014
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Bone tissue is continuously remodeled by bone-forming osteoblasts and bone-resorbing osteoclasts, in processes tightly regulated by hormones, cytokines and growth factors. CD47, a ubiquitously expressed protein, and one of its ligands, signal-regulatory protein alpha (SIRPα), are two cell-surface proteins belonging to the immunoglobulin (Ig)-superfamily. The interaction between CD47 and SIRPα is important for, amongst other processes, the fusion of macrophages into giant cells, which are closely related to osteoclasts.The aim of the present study was to gain knowledge about the role of CD47–SIRPα interaction and resultant downstream signaling pathways in bone cell differentiation, formation and function.The addition of antibodies against CD47 or SIRPα inhibited the formation of multinucleated osteoclasts from bone marrow monocytes (BMMs) in culture. Moreover, a significant decrease in the number of osteoclasts was detected in CD47-/- BMM cultures compared to CD47+/+ cultures. In line with these in vitro results, we found fewer osteoclasts in vivo in the trabecular bone of CD47-/- mice, as compared to CD47+/+ bone. Interestingly, an extended analysis of the trabecular bone of CD47-/- mice revealed that the bone volume, mineralizing surface, mineral apposition rate, bone formation rate and osteoblast number were also significantly reduced compared with CD47+/+ mice, indicating the importance of CD47 in osteoblast differentiation. In vitro studies of bone marrow stromal (BMS) cells from CD47-/- mice or SIRPα-mutant mice (mice lacking the signaling domain of SIRPa) showed a blunted expression of osteoblast-associated genes. Moreover, these altered genotypes were associated with reduced activity of the bone mineralization-associated enzyme alkaline phosphatase as well as a reduced ability to form mineral. To reveal the molecular mechanisms by which CD47 activation of SIRPα is important for BMS cell differentiation, we studied signaling downstream of SIRPα in the absence of CD47. In BMS cells lacking CD47, a considerable reduction in the levels of tyrosine phosphorylated SIRPα was detected, and the subsequent recruitment of the Src-homology-2 (SH2) domain-containing protein tyrosine phosphatase (SHP-2)–phosphoinositide 3-kinase (PI3K)–Akt2 signaling module was nearly abolished.In conclusion, the interaction between CD47 and SIRPα results in the activation of the SHP-2–PI3K–Akt2 pathway, which is necessary for normal osteoblast differentiation. In CD47-/- mice and SIRPα-mutant mice, this interaction is perturbed, which prevents normal osteoblast differentiation and subsequent mineral formation. In addition, the altered BMS cell phenotype results in an impaired ability to stimulate osteoclast differentiation.
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| 4. |
- Lindmark, Maria, 1969-
(författare)
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Regulation of phagocytosis and phagolysosome fusion in human leukocytes
- 2003
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Professional phagocytes such as neutrophil granulocytes and macrophages are an esential part of the innate immune system. The neutrophils form the first line of defence against invading microorganisms and are important for rapid killing of the intruders. Macrophages arrive later at the site of infection, kill micoorganisms, degrade dead cells, present antigens and secrete substances that orchestrate the inflammatory response. Neutrophils and macrophages ingest and kill microorganisms in a process called phagocytosis, where calcium signalling has shown to be involved. Inside the cell the microorganism is enclosed in a phagosome, that sequentially fuses with various intracellular vesicles to form a phagolysosome in which the intruder is killed. Killing is achieved through the actions of lytic enzymes, nitrogen oxide (NO) and reactive oxygen metabolites (ROM). Studies on the regulation of phagocytosis are essential since many pathogens are able to survive by interfering with this process. In the first study we investigated intracellular signalling in human neutrophils following engagement of a phagocytic receptor, complement receptor 3 (CR3). For this, we used antibody-coated PANSORBINS® which bound to the ß-chain of CR3 without inducing phagocytosis. We found that these particles elicited an intracellular production of ROM which was dependent on the cytoskeleton and on phospholipase D. In the second study, we showed that the putative calcium-sensor synaptotagmin II is present in neutrophils and is involved in phagocytosis. Synaptotagmin II was found on the specific granules and translocated to the phagosome in a calcium-dependent manner during eR-mediated phagocytosis and to the plasma membrane after stimulation with the formylated peptide, N-formyl-methionyl-leucyl-phenylalanine. In the third study, we demonstrate the presence of synaptotagmin IV in human macrophages. Synaptotagmin IV translocated transiendy to macrophage phagosomes during eR- and FcγR-mediated phagocytosis. We also found that eR- and FcyR-mediated uptake was calcium dependent in these cells. In the fourth study, we show that lipophosphoglycan (LPG) from Leishmania donovani induced elevated levels of periphagosomal F-actin, inhibition of phagolysosome maturation and diminished production of ROM in neutrophils during eR-mediated phagocytosis. Together, our data show that generation of ROM occurs early during eR-mediated phagocytosis and could be involved in intracellular signalling, that synaptotagmins are present in professional phagocytes and could act as calcium sensors in phagosomal maturation and secretion, and that LPG can be used as a tool to investigate how actin can regulate phagosomal maturation in neutrophils.
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| 5. |
- Olsson, Mattias, 1975-
(författare)
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Role of the CD47/SIRPα-interaction in regulation of macrophage phagocytosis
- 2008
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- CD47 is a cell surface glycoprotein that is expressed by virtually all cells in the body. Binding of CD47 to the macrophage receptor Signal Regulatory Protein alpha (SIRPα) yields an inhibitory signal that counteracts phagocytosis. Red blood cells (RBCs) that lack CD47 are rapidly cleared from the circulation, whereas CD47 expressing cells have a normal turnover rate. CD47 has therefore been proposed to function as a marker of self, enabling the immune system to discriminate between self and foreign. Thus, the studies of the present thesis aimed at further investigating the role of CD47 as a marker of self in regulating phagocytosis of platelets, phagocytosis of viable or senescent RBCs, and the mechanisms involved. CD47 on platelets was found to regulate their turnover in vivo, since platelets from CD47-/- mice transfused into wild type recipients were cleared more rapidly from the circulation than wild type platelets. In addition, CD47-/- mice were found to suffer from a mild spontaneous thrombocytopenia, without any signs of accelerated platelet apoptosis or increased platelet activation. CD47-/- mice were more sensitive to experimental immune thrombocytopenia (ITP), as compared with wild type mice. In vitro phagocytosis experiments proved that platelet CD47 was responsible for this effect, since blocking antibodies to macrophage SIRPα increased phagocytosis of wild type platelets to the levels seen for CD47-/- platelets. When unopsonized platelets or RBCs from CD47+/- mice (expressing about 50 % less CD47 than wild type cells) were transfused into wild type recipients, they were cleared from the circulation at virtually the same rate as wild type cells. However, CD47+/- cells were cleared more rapidly than wild type cells when transfused animals were challenged with an antibody directed against the transfused cell type. In vitro, IgG-opsonized CD47+/- platelets and RBCs were ingested to a higher extent than wild type cells, but less than CD47-/- cells, suggesting that CD47 dose-dependently regulates phagocytosis in macrophages. It was also investigated if inhibitory SIRPα signaling is localized to the site of contact with the cell that is to be ingested, or whether the inhibition of phagocytosis is more general in the whole macrophage. Experiments with a mix of IgG-opsonized wild type and CD47-/- RBCs showed that the effect of inhibitory CD47-SIRPα signaling was local in the macrophage and limited to the site of contact with a specific target cell. Thus, contact with one or several wild type RBCs did not affect the increased phagocytosis of CD47-/- RBCs by the same macrophage. RBC senescence involves oxidation of membrane lipids and proteins, as well as exposure of phosphatidylserine (PS) on the cell surface, and clearance of senescent RBCs is believed to be regulated by several different factors. To investigate the role of CD47 in uptake of experimentally senescent RBCs, RBCs were oxidized with CuSO4/ascorbic acid (Ox-RBCs). Phagocytosis of Ox-RBCs required recognition of PS on the RBCs, recognition by scavenger receptors on the macrophages, and was strongly dependent on serum. CD47 did not inhibit serum-dependent phagocytosis of experimentally senescent unopsonized RBCs, since phagocytosis of senescent wild type or CD47-/- RBCs was virtually similar. The ability of CD47 to cluster in the plasma membrane upon cross-linking with antibodies was reduced in senescent RBCs. Despite this, CD47 inhibited phagocytosis of IgG-opsonized viable or senescent RBCs to the same extent. In summary, CD47 can function as a marker of self on both RBCs and platelets. The phagocytosis-inhibitory effect is dependent on the CD47 expression level, and CD47-SIRPα signaling acts locally in the macrophage at the contact with a target cell. In experimentally senescent RBCs, CD47 does not inhibit serum-dependent phagocytosis in the absence of opsonization, but still inhibits FcγR-mediated phagocytosis. Key words: CD47, SIRPα, platelets, red blood cells, macrophages, phagocytosis, Fcγ receptor, senescence
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| 6. |
- Persson, Alexander, 1978-
(författare)
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Apoptotic neutrophils enhance the immune response against Mycobacterium tuberculosis
- 2009
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis, a disease that for years was considered to belong of the past, but tuberculosis is back causing over 2 million deaths per year. The infection can be dormant for decades and an active immune response can prevent the infection from progressing into active disease. However, the HIV/AIDS epidemic has caused an alarming rise in tuberculosis cases.The main infectious route for Mtb is through the airways into the lungs, where they encounter alveolar macrophages. Mtb are phagocytosed by these macrophages, but instead of being killing within the phagosome, Mtb modulates the cell to become a host in which the bacteria thrive. The lack of capacity to eradicate the infection stimulate cells of the immune system to gather around infected macrophages and form a granuloma that walls off the infection. Within this granuloma, Mtb can wait silently and later progress into active disease. However, only a fraction of exposed individuals develop disease, indicating that initial eradication of Mtb infections is possible. Such immediate response must be directed by the innate immunity comprised of phagocytes such as neutrophils (PMNs) and non-activated macrophages. Upon Mtb infection, macrophages become anergic and PMNs enter apoptosis. PMNs have a short lifespan and are cleared by neighbouring phagocytes, a mechanism described to resolve the inflammation and modulate tissue regeneration.We found that Mtb-induced apoptosis in PMNs was not dependent on phagocytosis of the bacteria, indicating that Mtb have the capacity to induce apoptosis in multiple PMNs. Complement-mediated phagocytosis induce survival signals such as Akt in PMNs, but despite this, complement-opsonized Mtb was able to override the anti-apoptotic activation in the cells. Since phagocytes clear apoptotic cells, we investigated how clearance of Mtb-induced apoptotic PMNs affected macrophages. We found that Mtb-induced apoptotic PMNs inflicted pro-inflammatory activation of the macrophages that cleared them. In addition, this activation was mediated by Hsp72 released from the Mtb-induced apoptotic PMNs. Furthermore, apoptotic PMNs can work in synergy with phagocytosed Mtb to activate macrophages and enhance intracellular killing of Mtb.Since dendritic cells are important for the regulation of immunity, we investigated whether Mtb-induced apoptotic PMNs affected the inflammatory response and maturation of dendritic cells. We found that Mtb-induced apoptotic PMNs trigger dendritic cells to enter a mature state able to activate naïve T-cell proliferation.We propose that infected apoptotic PMNs is a potent activator of the inflammatory response during infections. Taken together, PMNs not only kill their share of pathogens but also modulate other immune cells, thereby forming a link between the early innate and the adaptive immune response during microbial challenge with Mtb.
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| 7. |
- Saiepour, Daniel, 1976-
(författare)
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Glucose and insulin modulate phagocytosis and production of reactive oxygen metabolites in human neutrophil granulocytes
- 2006
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Neutrophil granulocytes play an important role in the host defence against invading microorganisms and constitute the frontline of defence within the innate immune system and are among the first cells to arrive at the site of inflammation. Effective phagocytosis and killing of invading pathogens by neutrophils is of significant importance for successful resistance to infectious diseases. An important complication in diabetes mellitus is an increased sensitivity to infections and increased tissue damage, leading to many secondary diseases. This may in part be explained by an impaired function of neutrophil granulocytes. Since the exact mechanisms underlying defective neutrophil function in diabetes mellitus are not fully understood, the aim of the present study was to investigate the effects of elevated glucose and insulin concentrations on phagocytosis of opsonized yeast and on production of reactive oxygen metabolites (ROS) in normal human neutrophils. Elevated D-glucose concentrations (15-25 mM) inhibited the phagocytosis of C3bi- or IgG-opsonized yeast particles, which was neither an osmotic effect nor an effect due to reduced binding of opsonized yeast particles to the neutrophils. Inhibition of protein kinase C (PKC) by GF109203X or Go6976 could completely reverse the inhibitory effect of 25 mM D-glucose on phagocytosis. Diacylglycerol (DAG) dose-dependently inhibited phagocytosis and suboptimal inhibitory concentrations of DAG and glucose showed an additive inhibitory effect. Elevated concentrations of insulin (80-160 μU/ml) also inhibited neutrophil phagocytosis, an effect shown in part to be due to a delayed phagocytosis process. Insulin was found to increase the accumulation of cortical F-actin, without affecting the total cellular F-actin content. The PKCalpha/beta inhibitor, Go6976, abolished the insulin-mediated increase in cortical F-actin content and both Go6976 and the PKCalpha/beta/delta/epsilon-specific inhibitor GF109203X reversed the inhibitory effects of insulin on phagocytosis. The inhibition of phagocytosis by either glucose or insulin resulted in an expected reduction of intracellular respiratory burst. However, the extracellular release of ROS during phagocytosis was increased by insulin, but inhibited by glucose. The ability of insulin to enhance ROS production was found to be F-actin dependent. Data suggests that glucose inhibited intracellular respiratory burst activation by interfering with intracellular signaling downstream of PKC activation, whereas extracellular release of ROS was inhibited by glucose upstream of PKC signaling. Taken together these results suggest that both hyperglycemia and hyperinsulinemia inhibit complement receptor and Fc receptor-mediated phagocytosis in human neutrophils. Insulin, but not glucose, also induced an enhanced extracellular release of ROS during phagocytosis. The combination of reduced phagocytosis and alterations in ROS production may possibly explain both the increased sensitivity to infections and tissue damage seen in type 2 diabetes.
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| 8. |
- Thyagarajan, Radha, 1986-
(författare)
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Anomalies in humoral immunity in the NOD mouse : contribution to the progression of type 1 diabetes
- 2016
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The non-obese diabetic (NOD) mouse is widely used model Type 1 diabetes (T1D), a chronic inflammatory disease characterized by destruction of the insulin producing β cells in the islets of Langerhans by immune cells. The classical symptoms include increased glucose levels in urine and blood, frequent urination and enhanced thirst. The disease has a strong genetic component and is also influenced by the environment. NOD mice develop T1D spontaneously. The disease occurs in two phases; insulitis - the infiltration of immune cells in the islets of Langerhans and overt diabetes caused by the destruction of insulin producing β cells. Several disease associated gene regions or loci [termed insulin dependent diabetes (Idd) loci] have been associated with T1D development. Although, T1D is recognized as a T cell mediated disease in both mouse and man, many studies have shown the importance of B cells in the pathogenesis of the disease. Autoantibodies appear prior to islet infiltration and several molecular and cellular events precede this beta-cell autoimmunity. Although the pathogenesis of T1D is well characterized, less is known about the environmental and immunological factors that trigger the disease.In this thesis, we studied the contribution of B cell anomalies to the skewed immune response observed in the NOD mouse. In our studies covered in the thesis we observed that NOD mice display enhanced IgE in the serum already at one week of age. In addition, upon treatment of pre-diabetic NOD mice with anti-IgE antibodies, diabetes incidence was delayed. We hypothesize that the presence of IgE in the system may be explained due to enhanced class switching. Antibody feedback however, is an essential component of the immune response and can lead to either enhanced or dampened responses. Thus, increased IgE may provide positive feedback that might sustain an immune response. We also aimed to analyze the biological consequence of this feature. In vitro stimulation of B cells by the TACI ligand APRIL resulted in enhanced plasma cell differentiation accompanied with increased class switching and IgG production. In addition, TACI+ cells were observed in NOD germinal centers facilitating increased BAFF uptake and subsequent escape of low affinity antibody producing clones. NOD mice elicited an enhanced and prolonged immune response towards T-dependent antigens such as hen-egg lysozyme (HEL). Serum HEL-specific IgG level was significantly increased and was predominantly of the IgG1 isotype. Immunofluorescence analysis of NOD spleen revealed the presence of spontaneous germinal centers which others have perceived to provide a ready niche for the entry of naïve B cells that encountered novel antigen. Adoptive transfer experiments of purified B and T cells from NOD into NOD.Rag2-/- (NOD-RAG) mice illustrated the importance of B cell intrinsic defects in the reproduction of the original phenotype as observed in NOD.
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